Selective enrichment medium for carbapenem-resistant bacteria

ABSTRACT

The present invention relates to a method for direct detection and differentiation of carbapenem-resistant bacteria in a sample comprising (i) inoculation with said sample of a culture medium comprising at least meropenem and/or ertapenem and at least one chromogenic agent, (ii) incubation of said culture medium under conditions allowing the growth of carbapenem-resistant bacteria, and (iii) detection of colonies formed on said culture medium corresponding to a carbapenem-resistant bacteria, as well as a culture medium suitable for use in such a method.

FIELD OF THE INVENTION

The present invention relates to a method for specific and directdetection of carbapenem-resistant bacteria in a sample using a selectivedifferential isolation medium for resistant bacteria comprising at leastone carbapenem.

PRIOR ART

The carbapenems are a category of antibiotics obtained from Streptomycescattleya. This is a family of antibiotics that is effective at lowconcentrations against the majority of Gram (−) bacteria, as well asagainst Gram (+) and anaerobic bacteria, including practically allbeta-lactamase producing strains.

Various publications have reported the emergence of resistance tocarbapenems in a wide variety of Gram (−) bacteria including Pseudomonasaeruginosa, Burkholderia cepacia, Acinetobacter species, Proteusspecies, Serratia marcescens, Enterobacter species and Klebsiellapneumoniae. This resistance to carbapenems can be highly transmissible.

Obviously, such resistance has serious implications for the treatment ofbacterial infections with antibiotics from the carbapenem family insofaras bacterial resistance can lead to the failure of these treatments andepidemic transmission of this resistance. Identification of patientsamples showing resistance to carbapenems and differentiation of variousresistant strains is therefore particularly important because it canhelp limit the risk of treating a patient with unsuitable antibiotics.Thus, the risk of propagation of the infection can be limited by meansof isolation techniques of infected patients and confinement of thesestrains by healthcare staff (special cleaning, use of gloves, dedicatedmedical materials and hospitalisation).

It is therefore important to have available detection tools and methodsfor these bacteria which combine both good specificity and selectivityand, most importantly, ease of use such that tests can be simplified asmuch as possible, carried out quickly and in large numbers, and possiblyeven automated, in order to control hospital hygiene with regard tobacteria posing a nosocomial risk, at the same time as allowing rapiddifferentiation between various strains which might be resistant tocarbapenems.

In the prior art, a microdilution technique was widely used for thedetection of carbapenem-resistant bacteria but this is currently underquestion in view of the number of false positive results to imipenemfound by several studies (CARMELI et al., Journal of ClinicalMicrobiology, vol. 36, p:595-597, 1998; GRIST, Journal of ClinicalMicrobiology 30:535-536, 1992). Given this, the technique that mainlyused at the present time is that of diffusion discs which requires,similarly to microdilution, preliminary isolation of the strain whoseresistance or not to carbapenem is to be tested, thus requiring verytedious application within the scope of systematic hospital monitoring.

Overall, the current methods of detection consist of two successivesteps in order to allow specific detection of carbapenem-resistantbacteria: isolation on a non-selective and non-differential mediumfollowed by analysis of sensitivity to various antibiotics. This means afairly long period of time for screening and diagnosis, thus increasingthe risk of the spread of multi-resistant bacteria in treatment centresand the risk of error. Moreover, these detection methods do not make itpossible to differentiate between various strains which might beresistant to carbapenems.

Thus, there is presently a considerable need for a simple, morespecific, more direct, faster detection technique which also allowsvarious carbapenem-resistant bacterial strains to be differentiated butwhich does not require a combination of several consecutive tests,leading to further delays in obtaining results and increasing the riskof parasitic contamination or error as well as the risk of the spread ofmulti-resistant bacteria in treatment centres.

SUMMARY OF THE INVENTION

The present invention relates to a method for direct detection ofcarbapenem-resistant bacteria in a sample comprising the followingsuccessive steps:

a) Inoculation with said sample of a culture medium comprising at leastmeropenem and/or ertapenem and at least one chromogenic agent,

b) Incubation of said culture medium under conditions allowing thegrowth of carbapenem-resistant bacteria, and

c) Detection of colonies formed on said culture medium corresponding tocarbapenem-resistant bacteria.

Another object of the present invention relates to a culture mediumsuitable for use in said detection method.

DESCRIPTION OF THE INVENTION

Surprisingly and unexpectedly, the inventor has shown that use ofmeropenem and/or ertapenem in place of imipenem makes it possible, inchromogenic differential media, to limit false positive results for thedetection of carbapenem-resistant bacteria and to directly detectcarbapenem-resistant bacteria, at the same time as differentiatingbetween them and obtaining species identification information. Inparticular, when carried out on a solid agar medium, the detectionmethod developed by the inventor can be carried out directly on apatient sample without the need for a preliminary isolation step of thedifferent strains present in a sample.

In particular, the method of the invention can be applied to thedetection of carbapenem-resistant strains of the KPC+ bacteria typewhich carry the KPC gene.

Consequently, a first object of the present invention consists in amethod for the direct detection of carbapenem-resistant bacteria in asample comprising the following successive steps:

a) Inoculation with said sample of a culture medium comprising at leastmeropenem and/or ertapenem and at least one chromogenic agent,

b) Incubation of said culture medium under conditions allowing thegrowth of carbapenem-resistant bacteria, and

c) Detection of colonies formed on said culture medium corresponding tocarbapenem-resistant bacteria.

Advantageously, the method according to the present invention moreovercomprises a step d) allowing the presence or not of a particularbacterial strain to be ascertained as a function of the colour of thecolonies formed.

Compared to previous methods, the method developed by the inventorallows direct and rapid detection of carbapenem-resistant bacteria aswell as immediate differentiation of various carbapenem-resistantstrains by means of chromophore(s) released or not by the chromogen(s).In parallel, meropenem and ertapenem respectively show very good or goodstability in an agar medium (2 to 4 months) and the detection methodaccording to the invention has shown that, in such a medium, thedetection method can be carried out effectively over several months.

In fact, meropenem has shown an important and unexpected stability.

Preferably, the antibiotic used to apply the detection method ismeropenem. Generally, carbapenem-resistant bacteria belong to thefollowing genera: Pseudomonas (e.g., Pseudomonas aeruginosa),Burkholderia (e.g., Burkholderia cepacia), Acinetobacter, Proteus,Serratia (e.g., Serratia marcescens), Enterobacter, Klebsiella (e.g.,Klebsiella pneumoniae) or Escherischia (e.g., Escherischia coli).

Preferably, “carbapenem-resistant bacteria” means bacteria containing agene coding for a beta-lactamase which hydrolyses carbapenems of the KPCfamily (Klebsiella pneumoniae carbapenase) such as KPC-1 (e.g.,Klebsiella pneumoniae, access number: AAG13410), KPC-2 (e.g., Klebsiellapneumoniae, access number: AAK70220), KPC-3 (e.g., Klebsiellapneumoniae, access number: AAL05630), KPC-4 (e.g., Klebsiellapneumoniae, access number: ACA34343), KPC-5 (e.g., Pseudomonasaeruginosa, access number: ABY91240), KPC-6 (e.g., Klebsiellapneumoniae, access number: ACB71165).

The term “direct detection method” means a method which does not includea preliminary isolation step of the various bacterial strains present inthe sample, preferably a method which does not include a preliminaryisolation step of each of the bacterial strains present in the sample.

In fact, the method of the invention avoids the isolation step forbacterial colony candidates which can then undergo a more precise testto confirm their carbapenem-resistance status. It therefore applies tothe raw sample comprising a mixture of bacteria.

The term “sample” means a mixture of different bacterial strainsbelonging to different species, or even different genera. As an example,said sample corresponds to a mixture of at least two different bacterialstrains, preferably at least five different bacterial strains and, morepreferably, to at least ten different bacterial strains.

Said sample advantageously corresponds to a liquid biological samplesuch as saliva, blood or urine, or a solid sample such a faeces, orequally to a derivative of a liquid or solid biological sample (faeces,etc) such as a pre-culture of such a liquid or solid biological sample.

Advantageously, said sample is a liquid or solid biological sample.

The term “chromogenic agent” means a compound with a chromophorereleased after hydrolysis by a specific enzyme of a particular bacterialgenus or species.

The use of chromogenic agent in the medium permits to differentiatebetween various strains which might be resistant to carbapenems.

By “differentiate”, one should understand that the color(s) obtained bythe release or non-release of (a) chromophore(s) on the strain detectedon the medium permits to distinguish between various strains which mightbe resistant to carbapenems, as illustrated in the following exemples.

For example, the enzymes whose activity can be used within the scope ofthe present invention includes: β-D-glucuronidase or β-D-galactosidase,positive in Escherischia coli; β-D-glucosidase, positive in Klebsiella,Enterobacter, Serratia and Citrobacter; and/or desaminase, positive inProteus, Providencia and Morganella.

Preferably, said chromogenic agent(s) carries a chromophore releasedafter hydrolysis by a specific enzyme of a species or genus selectedfrom the group consisting of: Pseudomonas (e.g., Pseudomonasaeruginosa), Burkholderia (e.g., Burkholderia cepacia), Acinetobacter,Proteus, Serratia (e.g., Serratia marcescens), Enterobacter, Klebsiella(e.g., Klebsiella pneumoniae) and Escherischia (e.g., Escherischiacoli).

Alternatively, the chromogenic agents can be chosen to allowdifferentiation of different clones of the same species rather thanspecies or genus differentiation. As an example, chromogens can bechosen which make it possible to distinguish between differentKlebsiella pneumoniae clones which infect the same hospital, in order tocarry out epidemiological monitoring of these clones within the scope ofhospital hygiene and gradual eradication. With regard to thechromophores whose release is to be obtained by means of application ofone or more enzymatic activities of one or more strains of themicro-organisms to be detected, the following can be cited:O-nitrophenyl, P-nitrophenyl, ChloroNitrophenyl, Hydroxyphenyl,Nitroanilide, Phenolphthalein and Thymophthalein, Hydroxyquinoline,Cyclohexenoesculetine, Dihydroxyflavone, Catechol, Resazurin, Resofurin,VBzTM, VLM, VLPr, VQM, Indoxyl, 5-bromo-4-chloro-3-indoxyl,5-bromo-6-chloro-3-indoxyl, 6-chloro-3-indoxyl, 6-fluoro-3-indoxyl,5-lodo-3-indoxyl, N-Methylindoxyl, or other (refer to BIOSYNTH. orGLYCOSYNTH catalogue, www.biosynth.com and www.glycosynth.co.uk).

Preferably, the cenzymatic substrate of the chromogenic agent isselected in the group consisting in beta-D-glucopyranoside andbeta-D-galactopyranoside, or a combination thereof, and more preferablythe chromogenic agent is selected in the group consisting in1-O-Methyl-beta-D-glucopyranoside, o-nitrophenyl-beta-D-glucopyranoside, 5-bromo-4-chloro-3-indolyl-beta-D-glucopyranoside,4-methylumbelliferyl beta-D-galactopyranoside,5-bromo-6-chloro-3-indolyl beta-D-galactopyranoside,2-nitrophenyl-beta-D-galactopyranoside,4-nitrophenyl-beta-D-galactopyranoside, N-Methylindolyl-beta-D-galactopyranoside,5-iodo-3-indolyl-beta-D-galactopyranoside,beta-trifluoromethyl-umbelliferyl-beta-D-galactopyranoside andindolyl-beta-D-galactopyranoside.

Preferably, said culture medium is an agar culture medium, wherein theculture medium is, for example, agar-based.

Meropenem and ertapenem are well known compounds to the man skilled inthe art and can be obtained from MOLEKULA Ltd, for example.

The effective quantity of meropenem and/or ertapenem which is selectivefor carbapenem-resistant bacteria can be quite simply established by theman skilled in the art from his general knowledge and the resultsdescribed in the examples below.

Advantageously, meropenem concentration is between 0.25 and 2 mg/L,preferably between 0.3 and 1 mg/L and, and more preferably, between 0.35and 0.8 mg/L.

Advantageously, ertapenem concentration is between 0.5 and 2 mg/L,preferably between 0.8 and 1.5 mg/L and, and more particularly, between0.9 and 1.2 mg/L. The incubation conditions allowing the growth ofcarbapenem-resistant bacteria are well known to the man skilled in theart and are not different from the usual methods.

Moreover, the culture medium used in the method of the invention canadvantageously include at least one substance which reacts specificallywith a particular bacterial genus.

For example, the culture medium used in the method of the invention caninclude deoxycholate which kills Gram (+) strains, preferably at aconcentration of 0.8 to 1.2 g/L and/or amphotericin which kills yeasts,preferably at a concentration of 3 to 7 mg/L, and even more preferablyfrom 4 to 6 mg/L.

Preferably, the culture medium used in the method according to thepresent invention is a CHROMagar Orientation medium (BD DIAGNOSITCSYSTEM) comprising at least one antibiotic chosen from among meropenemand ertapenem or a combination thereof.

A second object of the invention relates to a culture medium likely tobe applied in a direct detection method of carbapenem-resistant bacteriain a sample such as that described previously.

Advantageously, said culture medium is an agar medium.

Advantageously, the antibiotic used in the culture medium of the presentinvention is meropenem.

A third object of the invention relates to the use of a culture mediumsuch as that described previously for the direct detection anddifferentiation of carbapenem-resistant bacteria.

The following examples are given for the purpose of illustration onlyand in no way limit the extent of the present invention.

EXAMPLES 1) Tests on Meropenem Versus Imipenem to SelectCarbapenem-Resistant Bacteria

Different strains of carbapenem-sensitive bacteria orcarbapenem-resistant bacteria were cultured under three sets ofconditions:

-   -   group 1: CHROMagar Orientation medium    -   group 2: CHROMagar Orientation medium in the presence of        meropenem (0.065; 0.25; 1 or 4 mg/L); and    -   group 3: CHROMagar Orientation medium in the presence of        imipenem (MSD, TIENAM (includes 50% imipenem) 0.0325; 0.125; 0.5        or 2 mg/L of imipenem)

The results show that the minimum concentration of meropenem (0.25 mg/L)to eliminate carbapenem-sensitive flora is about ten times lower thanthe necessary minimum concentration of imipenem (lmg/L).

2) Ageing Tests on Meropenem Versus Imipenem to SelectCarbapenem-Resistant Bacteria

Differents strains of carbapenem-sensitive bacteria orcarbapenem-resistant bacteria were cultured under three different setsof conditions and with different spreading concentrations:

-   -   group 1: CHROMagar Orientation medium    -   group 2: CHROMagar Orientation medium in the presence of        imipenem (MSD, TIENAM (includes 50% imipenem) 1 or 2 mg/L        imipenem), and    -   group 3: CHROMagar Orientation medium in the presence of        meropenem (0.12 or 0.25 mg/L).

Culture plates were then incubated at 37° C. and analysed regularly toestablish the growth or absence of growth of bacteria.

The results showed that at a concentration of 2 mg/L of imipenem,inhibition of the growth of carbapenem-sensitive bacteria after 55 daysin culture was similar to that obtained at a concentration of 1 mg/Lafter 21 days in culture. The results also confirmed that meropenem ismuch more effective than imipenem in terms of inhibition and that after55 days in culture, the inhibition of the growth of carbapenem-sensitivebacteria at 55 days in culture in the presence of 0.25 mg/L of meropenemis always higher than inhibition obtained at a concentration of 0.12mg/L of meropenem after 36 days in culture.

Finally, the results unexpectedly show that meropenem is a much moreeffective candidate for the selection of carbapenem-resistant bacteriathan imipenem, but more especially that meropenem also has much higherstability in the used culture medium compared to imipenem.

3) Tests on Ertapenem to Select Carbapenem-Resistant Bacteria

Different strains of carbapenem-sensitive bacteria orcarbapenem-resistant bacteria were cultured under two sets ofconditions:

-   -   group 1: CHROMagar Orientation medium; and    -   group 2: CHROMagar Orientation medium in the presence of        ertapenem (INVAZ; 0.0325; 0.065; 0.125; 0.25; 0.5 or 2 mg/L).

The results show that the minimum concentration needed to eliminatecarbapenem-sensitive flora falls between that of meropenem and imipenem.

4) Direct Detection of Carbapenem-Resistant Bacteria Using Meropenem

Various urine samples from patients suffering from bacterial infectionswere taken.

Said samples were added directly to Petri dishes with CHROMagarOrientation agar medium, which contains two chromogens substrates, i.e.beta-D-glucopyranoside and beta-D-galactopyranoside, in the presence of0.25 mg/L meropenem.

After incubation for 18 to 24 hours, visual analysis of the Petri dishesallowed direct identification of dishes containing bacteria.

The results are given in the table below:

Species Colour of colony E. coli Red Klebsiella Metallic blue

Thus, the method of the invention makes it possible, in a singleculturing step, to specifically detect and differentiatecarbapenem-resistant bacteria without the need for a preliminaryisolation step or subsequent differentiation step.

1. Method for direct detection of carbapenem-resistant bacteria in asample comprising the following successive steps: a) Inoculation withsaid sample of a culture medium comprising at least meropenem and/orertapenem and at least one chromogenic agent, b) Incubation of saidculture medium under conditions allowing the growth ofcarbapenem-resistant bacteria, and c) Detection of colonies formed onsaid culture medium corresponding to carbapenem-resistant bacteria. 2.Method according to claim 1, characterised in that thecarbapenem-resistant bacteria are selected from the group consisting ofthe following genera: Pseudomonas, Burkholderia, Acinetobacter, Proteus,Serratia, Enterobacter, et Klebsiella et Escherischia.
 3. Methodaccording to claim 1, characterised in that said bacteria are bacteriacontaining a gene coding for a beta-lactamase which hydrolysescarbapenems of the KPC Family (Klebsiella pneumoniae carbapenase). 4.Method according to claim 1, characterised in that said method does notinclude a preliminary isolation step of the different bacterial strainspresent in a sample.
 5. Method according to claim 1, characterised inthat said chromogenic agent is sensitive to the activity of at least oneenzyme selected in the group consisting of β-D-glucuronidase,β-galactosidase, β-D-glucosidase and a deaminase.
 6. Method according toclaim 1, characterised in that the chromophore released by enzymatichydrolysis of the chromogenic agent is chosen from among O-nitrophenyl,P-nitrophenyl, ChloroNitrophenyl, Hydroxyphenyl, Nitroanilide,Phenolphthalein, Thymophthalein, Hydroxyquinoline,Cyclohexenoesculetine, Dihydroxyflavone, Catechol, Resazurin, Resofurin,VBzTM, VLM, VLPr, VQM, Indoxyl, 5-bromo-4-chloro-3-indoxyl,5-bromo-6-chloro-3-indoxyl, 6-chloro-3-indoxyl, 6-fluoro-3-indoxyl,5-lodo-3-indoxyl and N-Methylindoxyl.
 7. Method according to claim 1,characterised in that said culture medium is an agar culture medium. 8.Method according to claim 1, characterised in that said culture mediumcontains meropenem at a concentration between 0.25 and 2 mg/L,preferably between 0.3 and 1 mg/L.
 9. Method according to claim 1,characterised in that said culture medium contains ertapenem at aconcentration between 0.5 and 2 mg/L, preferably between 0.9 and 1.2mg/L.
 10. Culture medium suitable for use in a detection method asdefined in claim
 1. 11. Culture medium according to claim 10,characterised in that the culture medium is an agar culture medium.